DNA double strand breaks (DSBs) are a particularly severe and mutagenic form of DNA damage as they disrupt genetic continuity. Proteins involved in prevention and repair of DSBs are associated with a variety of severe human diseases, such as breast and ovarian cancer (BRCA1 and BRCA2), Nijmegan breakage Syndrome (NBS1), Blood's syndrome (BLM), Ataxia telangiectasia (Mre11) and premature aging (WRN). In this project we aim to understand, at the molecular level, early events of DSB repair (DSBR) such as DSB recognition and processing, DSBR by non-homologous end joining (NHEJ), and DSB prevention by RecQ family helicases. Research of t he past years revealed that proteins involved in DSBR often not only interact with each other and other DNA repair proteins but exist in large super assemblies or repairosomes. The resulting complexity of protein-protein interactions and of conformational switches makes it necessary to shift efforts to understand the underlying molecular events, from the analysis of individual components, as has been successfully performed in the past, to the study of complexes and super-assemblies. For that reason, we propose to elucidate molecular events of protein-protein interactions, assembly states and conformational switching, for early events in DSBR. Tight synergies with Projects 4 (Homologous Recombination Repair) and 5 (Mismatch Repair interactions), plus collaborative efforts within this Program Project, will ensure the required experimental innovations, aided by advance technologies for protein expression and protein characterization techniques (EMB Core) and for the structure determination of larger complexes (SCB Core). We will begin to understand the complex nature of DSBR conformational switching and interactions by focusing on five Specific Aims: Aim 1 will study functional and structural switches in the NHEJ machinery in response to phosphorylation; Aim 2 will study the structure and function of the Rad50/BRCA1 interaction; Aim 3 will study the modulation of end processing of the Rad50/Mre11/Nbs1 complex by RPA; Aim 4 will study the structure and function of DSB preventive RecQ helicases; and Aim 5 will study early temporal and spatial interaction of DSBR factors by a chromatin immunoprecipitation. The anticipated combined outcome of the proposed five specific Aims will be a molecular picture of protein-protein interactions and functional states orchestrating early events o DSBR. This picture will provide the molecular foundation for a detailed understanding of human diseases and cancer predispositions linked to DSBR proteins.